The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.
Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.
Germain
D
D
Genetics Section, National Research Council of Canada, Montreal, Quebec.
Zollinger
L
L
Racine
C
C
Gossard
F
F
Dignard
D
D
Thomas
D Y
DY
Crine
P
P
Boileau
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006056:Golgi Apparatus / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011333:Pro-Opiomelanocortin / Q000235:genetics; D043484:Proprotein Convertases; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013381:Subtilisins; D014162:Transfection; D001615:beta-Endorphin / Q000378:metabolism; D008083:beta-Lipotropin / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D004247:DNA; D008083:beta-Lipotropin; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae